Several clonal strains of rat pituitary tumor cells (GH cells) synthesize and secrete different amounts of two hormones, prolactin (PRL) and growth hormone (GH). The synthesis of these proteins can be modulated by externally added physiological and pharmacological agents. Our primary objectives is understanding the mechanism(s) involved in the graded basal level production of prolactin (PRL) and the regulation of its synthesis by thyrotropin-releasing hormone (TRH), hydrocortisone (HC), estradiol (E2), 5-bromodeoxyuridine (BrdUrd), and by growth factors such as epidermal growth factor (EGF) in GH cells. Though the model cell system is not composed of normal cells, it still responds to physiological agents such as TRH, HC, E2, and EGF as does the normal pituitary gland. Since distinct cell types are believed to produce specific hormones in the normal pituitary gland, the GH subclones that produce PRL and GH provide a novel model system for understanding the mechanisms whereby neoplastic cells can express some of the strictly controlled genetic information of normal cells. The synthesis of PRL by a distinct type of cell in the normal pituitary gland represents a differentiated function. The GH subclones that produce vastly different amounts of PRL provide an ideal system for investigating the cellular mechanisms that lead to the regulated synthesis of such a cell-specific protein and thus provide a novel means to understand the process of cellular differentiation. Our results suggest that the graded levels of PRL synthesis by different GH-cell subclones at the basal state and after treatment with TRH or BrdUrd are due to altered transcription of the PRL-specific RNA sequences. The striking effect of BrdUrd on PRL synthesis is being utilized to obtain information on hormone receptor interactions with specific DNA binding sites. The recently observed BrdUrd-induced amplification of the PRL gene will help elucidate the mechanism of turning the switch "on" or "off" for the process of gene amplification in this system. As drug-induced amplification seems limited within the 20 kilobase segment within and around the PRL gene-coding sequence, these results suggest initiation and termination of such preferential DNA replication site(s) within this region that selectively respond to the drug BrdUrd. Identification of this site(s) will be of significant interest for the understanding of gene amplification in eukaryotic cells. (C)